Optimization of tumor-treating field therapy for triple-negative breast cancer cells in vitro via frequency modulation.

PURPOSE: Currently, tumor-treating field (TTField) therapy utilizes a single "optimal" frequency of electric fields to achieve maximal cell death in a targeted population of cells. However, because of differences in cell size, shape, and ploidy during mitosis, optimal electric field characteristics for universal maximal cell death may not exist. This study investigated the anti-mitotic effects of modulating electric field frequency as opposed to utilizing uniform electric fields. METHODS: We developed and validated a custom device that delivers a wide variety of electric field and treatment parameters including frequency modulation. We investigated the efficacy of frequency modulating tumor-treating fields on triple-negative breast cancer cells compared to human breast epithelial cells. RESULTS: We show that frequency-modulated (FM) TTFields are as selective at treating triple-negative breast cancer (TNBC) as uniform TTFields while having a greater efficacy for combating TNBC cell growth. TTField treatment at a mean frequency of 150 kHz with a frequency range of ± 10 kHz induced apoptosis in a greater number of TNBC cells after 24 h as compared to unmodulated treatment which led to further decreased cell viability after 48 h. Furthermore, all TNBC cells died after 72 h of FM treatment while cells that received unmodulated treatment were able to recover to cell number equivalent to the control. CONCLUSION: TTFields were highly efficacious against TNBC growth, FM TTFields showed minimal effects on epithelial cells similar to unmodulated treatment.

Efficacy and selectivity of tumor-treating field therapy for triple-negative breast cancer cells via in-house delivery device.

PURPOSE: Triple-negative breast cancer continues to be one of the leading causes of death in women, making up 7% of all cancer deaths. Tumor-treating electric fields are low-energy, low-frequency oscillating electric fields that induce an anti-proliferative effect on mitotic cells in glioblastoma multiforme, non-small cell lung cancer, and ovarian cancer. Little is known about effects of tumor-treating fields on triple-negative breast cancer and known research for tumor-treating fields only utilizes low (< 3 V/cm) electric field intensities. METHODS: We have developed an in-house field delivery device capable of high levels of customization to explore a much wider variety of electric field and treatment parameters. Furthermore, we investigated the selectivity of tumor-treating field treatment between triple-negative breast cancer and human breast epithelial cells. RESULTS: Tumor-treating fields show greatest efficacy against triple-negative breast cancer cell lines between 1 and 3 V/cm electric field intensities while having little effect on epithelial cells. CONCLUSION: These results provide a clear therapeutic window for tumor-treating field delivery to triple-negative breast cancer.

The Influence of the Normal Mammary Microenvironment on Breast Cancer Cells.

The tumor microenvironment is recognized as performing a critical role in tumor initiation, progression, and metastasis of many cancers, including breast cancer. The breast cancer microenvironment is a complex mixture of cells consisting of tumor cells, immune cells, fibroblasts, and vascular cells, as well as noncellular components, such as extracellular matrix and soluble products. The interactions between the tumor cells and the tumor microenvironment modulate tumor behavior and affect the responses of cancer patients to therapies. The interactions between tumor cells and the surrounding environment can include direct cell-to-cell contact or through intercellular signals over short and long distances. The intricate functions of the tumor microenvironment in breast cancer have led to increased research into the tumor microenvironment as a possible therapeutic target of breast cancer. Though expanded research has shown the clear importance of the tumor microenvironment, there is little focus on how normal mammary epithelial cells can affect breast cancer cells. Previous studies have shown the normal breast microenvironment can manipulate non-mammary stem cells and tumor-derived cancer stem cells to participate in normal mammary gland development. The tumorigenic cells lose their tumor-forming capacity and are "redirected" to divide into "normal", non-tumorigenic cells. This cellular behavior is "cancer cell redirection". This review will summarize the current literature on cancer cell redirection and the normal mammary microenvironment's influence on breast cancer cells.

Biomedical applications of tannic acid.

Tannic Acid (TA) is a naturally occurring antioxidant polyphenol that has gained popularity over the past decade in the field of biomedical research for its unique biochemical properties. Tannic acid, typically extracted from oak tree galls, has been used in many important historical applications. TA is a key component in vegetable tanning of leather, iron gall ink, red wines, and as a traditional medicine to treat a variety of maladies. The basis of TA utility is derived from its many hydroxyl groups and its affinity for forming hydrogen bonds with proteins and other biomolecules. Today, the study of TA has led to the development of many new pharmaceutical and biomedical applications. TA has been shown to reduce inflammation as an antioxidant, act as an antibiotic in common pathogenic bacterium, and induce apoptosis in several cancer types. TA has also displayed antiviral and antifungal activity. At certain concentrations, TA can be used to treat gastrointestinal disorders such as hemorrhoids and diarrhea, severe burns, and protect against neurodegenerative diseases. TA has also been utilized in biomaterials research as a natural crosslinking agent to improve mechanical properties of natural and synthetic hydrogels and polymers, while also imparting anti-inflammatory, antibacterial, and anticancer activity to the materials. TA has also been used to develop thin film coatings and nanoparticles for drug delivery. In all, TA is fascinating molecule with a wide variety of potential uses in pharmaceuticals, biomaterials applications, and drug delivery strategies.

Degradation and release of tannic acid from an injectable tissue regeneration bead matrix in vivo.

The development of multifunctional biomaterials as both tissue regeneration and drug delivery devices is currently a major focus in biomedical research. Tannic Acid (TA), a naturally occurring plant polyphenol, displays unique medicinal abilities as an antioxidant, an antibiotic, and as an anticancer agent. TA has applications in biomaterials acting as a crosslinker in polymer hydrogels improving thermal stability and mechanical properties. We have developed injectable cell seeded collagen beads crosslinked with TA for breast reconstruction and anticancer activity following lumpectomy. This study determined the longevity of the bead implants by establishing a degradation time line and TA release profile in vivo. Beads crosslinked with 0.1% TA and 1% TA were compared to observe the differences in TA concentration on degradation and release. We found collagen/TA beads degrade at similar rates in vivo, yet are resistant to complete degradation after 16 weeks. TA is released over time in vivo through diffusion and cellular activity. Changes in mechanical properties in collagen/TA beads before implantation to after 8 weeks in vivo also indicate loss of TA over a longer period of time. Elastic moduli decreased uniformly in both 0.1% and 1% TA beads. This study establishes that collagen/TA materials can act as a drug delivery system, rapidly releasing TA within the first week following implantation. However, the beads retain TA long term allowing them to resist degradation and remain in situ acting as a cell scaffold and tissue filler. This confirms its potential use as an anticancer and minimally invasive breast reconstructive device following lumpectomy.

Asymmetric cell division of mammary stem cells.

Somatic stem cells are distinguished by their capacity to regenerate themselves and also to produce daughter cells that will differentiate. Self-renewal is achieved through the process of asymmetric cell division which helps to sustain tissue morphogenesis as well as maintain homeostasis. Asymmetric cell division results in the development of two daughter cells with different fates after a single mitosis. Only one daughter cell maintains "stemness" while the other differentiates and achieves a non-stem cell fate. Stem cells also have the capacity to undergo symmetric division of cells that results in the development of two daughter cells which are identical. Symmetric division results in the expansion of the stem cell population. Imbalances and deregulations in these processes can result in diseases such as cancer. Adult mammary stem cells (MaSCs) are a group of cells that play a critical role in the expansion of the mammary gland during puberty and any subsequent pregnancies. Furthermore, given the relatively long lifespans and their capability to undergo self-renewal, adult stem cells have been suggested as ideal candidates for transformation events that lead to the development of cancer. With the possibility that MaSCs can act as the source cells for distinct breast cancer types; understanding their regulation is an important field of research. In this review, we discuss asymmetric cell division in breast/mammary stem cells and implications on further research. We focus on the background history of asymmetric cell division, asymmetric cell division monitoring techniques, identified molecular mechanisms of asymmetric stem cell division, and the role asymmetric cell division may play in breast cancer.

Induction of phenotypic changes in HER2-postive breast cancer cells in vivo and in vitro.

The influence of breast cancer cells on normal cells of the microenvironment, such as fibroblasts and macrophages, has been heavily studied but the influence of normal epithelial cells on breast cancer cells has not. Here using in vivo and in vitro models we demonstrate the impact epithelial cells and the mammary microenvironment can exert on breast cancer cells. Under specific conditions, signals that originate in epithelial cells can induce phenotypic and genotypic changes in cancer cells. We have termed this phenomenon "cancer cell redirection." Once breast cancer cells are redirected, either in vivo or in vitro, they lose their tumor forming capacity and undergo a genetic expression profile shift away from one that supports a cancer profile towards one that supports a non-tumorigenic epithelial profile. These findings indicate that epithelial cells and the normal microenvironment influence breast cancer cells and that under certain circumstances restrict proliferation of tumorigenic cells.

Redirecting Normal and Cancer Stem Cells to a Mammary Epithelial Cell Fate.

Tissue microenvironments, also known as stem cell niches, influence not only resident cells but also cells in surrounding tissues. Physical and biochemical intercellular signals originating from resident stem cells or non-stem cells participate in the homeostasis of the tissue regulating cell proliferation, differentiation, wound healing, tissue remodeling, and tumorigenesis. In recent publications it has been demonstrated that the normal mouse mammary microenvironment can provide development and differentiation guidance to not only resident mammary cells but also cells of non-mammary origin including tumor-derived cells. When placed in reforming mammary stem cell niches the non-mammary cells proliferate and differentiate along mammary epithelial cell lineages and contribute progeny to reforming mammary gland outgrowths. The tumor-derived cells that are redirected to assume mammary epithelial phenotypes lose their cancer-forming capacity and shift their gene expression profiles from a cancer profile towards a normal mammary epithelial expression profile. This review summarizes the recent discoveries regarding the ability of the normal mouse mammary microenvironment to dictate the cell fates of non-mammary cells introduced into mammary stem cell niches.

Effects of Astaxanthin on the Proliferation and Migration of Breast Cancer Cells In Vitro.

Astaxanthin (ASX) is a marine-based ketocarotenoid; an accessory pigment in plants in that it has many different potential functions. ASX is an antioxidant that is notably more potent than many other antioxidants. Antioxidants have anti-inflammatory and oxidative stress-reducing properties to potentially reduce the incidence of cancer or inhibit the expansion of tumor cells. In this study, we tested the hypothesis that ASX would inhibit proliferation and migration of breast cancer cells in vitro. We found that application of ASX significantly reduced proliferation rates and inhibited breast cancer cell migration compared to control normal breast epithelial cells. Based on these results, further investigation of the effects of ASX on not only breast cancer cells, but other forms of tumor cells, should be carried out.

HER2+ breast cancer cells undergo apoptosis upon exposure to tannic acid released from remodeled cross-linked collagen type I.

Tannic acid (TA) is a naturally occurring polyphenol that cross-links collagen type I and possesses anticancer potential. In previous studies, we demonstrated the increased sensitivity of estrogen receptor-positive (ER+ ) breast cancer cells to TA as opposed to triple negative breast cancer cells and normal human breast epithelial cells. In the current study, human pre-adipocytes and HER2+ breast cancer cells were grown on TA cross-linked collagen type I beads. Cell attachment, growth, and proliferation of the cells result in remodeling of the collagen matrix and release of the cross-linking TA. TA concentrations in the conditioned media were determined. Induced apoptosis of cells grown on the TA cross-linked collagen type I beads was imaged and quantified. Viability of HER2+ breast cancer cells and normal breast epithelial cells after exposure TA released from bead remodeling was quantified. Caspase gene expression and protein expression were evaluated. HER2+ breast cancer cells underwent caspase-mediated apoptosis in response to TA exposure. TA-induced apoptosis in a concentration- and time-dependent manner, with HER2+ breast cancer cells demonstrating an increased sensitivity to the TA effects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 26-32, 2018.

Amphiregulin regulates proliferation and migration of HER2-positive breast cancer cells.

PURPOSE: Tumor initiation and progression rely on cellular proliferation and migration. Many factors are involved in these processes, including growth factors. Amphiregulin (AREG) is involved in normal mammary development and the development of estrogen receptor (ER)-positive breast cancer. The aim of this project was to determine if AREG is involved in the proliferation and progression of HER2-positive breast cancer. METHODS: Mouse cell lines MMTV-neu, HC-11 and COMMA-D, as well as human cell lines MCF10A, SKBR3, HCC1954 and BT474 were used. Real-time PCR was used to quantify AREG expression and neutralizing antibodies were used to reduce the autocrine/paracrine effects of AREG. Transfections using siRNA and shRNA were used to knockdown AREG expression in the cancer cell lines. Free-floating sphere formation, colony forming, scratch wound and Transwell assays were used to assess the proliferation, tumor forming and migratory capacities of transfected cancer cells. RESULTS: We found AREG expression in both normal epithelial cell lines and tumor-derived cell lines. Knockdown of AREG protein expression resulted in reduced sphere sizes and reduced sphere numbers in both mouse and human cancer cells that overexpress erbB2/HER2. AREG was found to be involved in cancer cell migration and invasion. In addition, we found that AREG expression knockdown resulted in different migration capacities in normal and erbB2/HER2 overexpressing cancer cells. CONCLUSIONS: Based on our results we conclude that AREG is involved in regulating the proliferation and migration of erbB2/HER2-positive breast cancer cells.

Cancer cell redirection biomarker discovery using a mutual information approach.

Introducing tumor-derived cells into normal mammary stem cell niches at a sufficiently high ratio of normal to tumorous cells causes those tumor cells to undergo a change to normal mammary phenotype and yield normal mammary progeny. This phenomenon has been termed cancer cell redirection. We have developed an in vitro model that mimics in vivo redirection of cancer cells by the normal mammary microenvironment. Using the RNA profiling data from this cellular model, we examined high-level characteristics of the normal, redirected, and tumor transcriptomes and found the global expression profiles clearly distinguish the three expression states. To identify potential redirection biomarkers that cause the redirected state to shift toward the normal expression pattern, we used mutual information relationships between normal, redirected, and tumor cell groups. Mutual information relationship analysis reduced a dataset of over 35,000 gene expression measurements spread over 13,000 curated gene sets to a set of 20 significant molecular signatures totaling 906 unique loci. Several of these molecular signatures are hallmark drivers of the tumor state. Using differential expression as a guide, we further refined the gene set to 120 core redirection biomarker genes. The expression levels of these core biomarkers are sufficient to make the normal and redirected gene expression states indistinguishable from each other but radically different from the tumor state.

RNA Expression Profiling Reveals Differentially Regulated Growth Factor and Receptor Expression in Redirected Cancer Cells.

Tumorigenic cells can be redirected to adopt a normal phenotype when transplanted into cleared mammary fat pads of juvenile female mice in specific ratios with normal epithelial cells. The redirected tumorigenic cells enter stem cell niches and provide progeny that differentiate into all mammary epithelial subtypes. We have developed an in vitro model that mimics the in vivo phenomenon. The shift in phenotype to redirection should be accomplished through a return to a normal gene expression state. To measure this shift, we interrogated the transcriptome of various in vitro model states in search for casual genes. For this study, expression of growth factors, cytokines, and their associated receptors was examined. In all, we queried 251 growth factor and cytokine-related genes. We found numerous growth factor and cytokine genes whose expression levels switched from expression levels seen in cancer cells to expression levels observed in normal cells. The comparisons of gene expression between normal mammary epithelial cells, tumor-derived cells, and redirected cancer cells have revealed insight into active and inactive growth factors and cytokines in cancer cell redirection.

Validation of an in vitro model of erbB2(+) cancer cell redirection.

Overexpression of the oncoprotein erbB2/HER2 is present in 20-30% of breast cancer patients and inversely correlates with patient survival. Reports have demonstrated the deterministic power of the mammary microenvironment where the normal mammary microenvironment redirects cells of non-mammary origin or tumor-derived cells to adopt a mammary phenotype in an in vivo model. This phenomenon is termed tumor cell redirection. Tumor-derived cells that overexpress the erbB2 oncoprotein lose their tumor-forming capacity in this model. In this model, phosphorylation of erbB2 is attenuated thus reducing the tumor cell's tumor-forming potential. In this report, we describe our results using an in vitro model based on the in vivo model mentioned previously. Tumor-derived cells are mixed in predetermined ratios with normal mammary epithelial cells prior to seeding in vitro. In this in vitro model, the tumor-derived cells are redirected as determined by attenuated phosphorylation of the receptor and reduced sphere and colony formation. These results match those observed in the in vivo model. This in vitro model will allow expanded experimental options in the future to determine additional aspects of tumor cell redirection that can be translated to other types of cancer.

Remodeling of tannic acid crosslinked collagen type I induces apoptosis in ER+ breast cancer cells.

BACKGROUND: The naturally-occurring phytochemical tannic acid (TA) has anticancer properties. We have demonstrated that estrogen receptor-positive (ER+) breast cancer cells are more sensitive to effects of TA than triple-negative breast cancer cells and normal breast epithelial cells. In the present study, cells were grown on TA-crosslinked collagen beads. Growing cells remodel collagen and release TA, which affects attached cells. MATERIALS AND METHODS: The ER+ breast cancer cell line MCF7 and the normal breast epithelial cell line MCF10A were grown on TA-crosslinked collagen beads in roller bottles. Concentrations of TA in conditioned media were determined. Induced apoptosis was imaged and quantified. Caspase gene expression was calculated by real-time polymerase chain reaction (PCR). RESULTS: Both cell lines attached and grew on TA-crosslinked collagen beads where they remodeled collagen and released TA into surrounding medium. Released TA induced caspase-mediated apoptosis. CONCLUSION: TA induced apoptosis in a concentration-dependent manner, with ER+ MCF7 cells displaying more sensitivity to effects of TA.

Tannic Acid preferentially targets estrogen receptor-positive breast cancer.

Research efforts investigating the potential of natural compounds in the fight against cancer are growing. Tannic acid (TA) belongs to the class of hydrolysable tannins and is found in numerous plants and foods. TA is a potent collagen cross-linking agent; the purpose of this study was to generate TA-cross-linked beads and assess the effects on breast cancer cell growth. Collagen beads were stable at body temperature following crosslinking. Exposure to collagen beads with higher levels of TA inhibited proliferation and induced apoptosis in normal and cancer cells. TA-induced apoptosis involved activation of caspase 3/7 and caspase 9 but not caspase 8. Breast cancer cells expressing the estrogen receptor were more susceptible to the effects of TA. Taken together the results suggest that TA has the potential to become an anti-ER(+) breast cancer treatment or preventative agent.

Evaluation of normal and metastatic mammary cells grown in different biomaterial matrices: establishing potential tissue test systems.

The in vitro growth and differentiation of normal mammalian cells is quite different than the growth of cells derived from tumors. Additionally, cells of the same origin (tissue) behave differently depending on the biomaterial matrix in or on which they are grown in vitro. We examined both Matrigel(TM) and a collagen/agarose blend and demonstrated that two murine mammary derived cells lines, 4T1 and NMuMG, derived from a metastatic mammary tumor or a normal mammary gland, respectively, exhibit different growth and differentiation patterns depending on the three-dimensional matrix in which they are grown. The shape and size of the colonies that formed were matrix dependent. The two cell lines produced different levels of growth factors and metalloproteinases, and expressed differentiation markers specific to a matrix. Through the classification of different cell behaviors in different growth matrices, we will be able to intelligently design and tune tissue test systems to ask and answer specific challenging scientific questions.

Differential gene expression in nuclear label-retaining cells in the developing mouse mammary gland.

The immortal strand theory postulates stem cells protect themselves from DNA replication-associated mutations and subsequent cancer risk through selective segregation of template DNA strands. Stem cells self-renew by asymmetric cellular division. During asymmetric division, stem cells maintain their template DNA strands, while the newly synthesized DNA strands segregate to newly formed daughter cells. Previous studies have demonstrated that self-renewing mammary stem cells originate in the expanding mammary ducts during puberty-associated allometric growth. In this study, we labeled newly forming mammary stem cells with the thymidine analog 5-ethynl-2'-deoxyuridine for 2 weeks during allometric ductal expansion. Cells that incorporate and retain the nuclear label following extended chase periods are termed label-retaining cells (LRCs). A second nuclear label, 5-bromodeoxyuridine, was administered before euthanasia to identify cells traversing the cell cycle. Mammary cells collected following euthanasia were sorted based on nuclear label retention. Members of the Notch and Wnt signaling pathways were found differentially expressed by mammary LRCs. These pathways are involved in the regulation of stem cells in the mouse mammary gland. Upon further analysis, we found that in contrast to non-LRCs, Notch1 and Notch2 are expressed and localized in the nuclei of the LRCs. Expression of Notch-inducible genes, Hes1 and Hey2, was elevated in LRCs. Inhibition of Notch1 by shRNA reduced colony forming potential and label retention by mammary epithelial cells in vitro. These results indicate that genes are differentially regulated in the LRC population of mammary glands and Notch1 mediates asymmetric cell division of mammary progenitor cells.